Determination of DPP-IV inhibition

Dipeptidyl-peptidase IV (DPP-IV) inhibitory activity was assessed according to the method described by Zeng et al. (12) with some modifications. Briefly, 25 μL Gly-Pro-p-nitroanilide (6 mM; Sigma-Aldrich) and 25 μL Tartary buckwheat sample, or 25 μL phosphate buffer saline (PBS; Sigma-Aldrich) as a control, were mixed and preincubated at 37 °C for 10 min. The reaction was initiated by adding 50 μL DPP-IV from porcine kidney (3·10^–4 U/L, ≥10 U/mg protein; Sigma-Aldrich) and the mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 100 μL of 1 M sodium acetate buffer (pH=4.0; Sinopharm Chemical Reagent Co., Ltd), and the absorbance of the samples at 405 nm was measured on a Multiskan MK3 plate reader (Thermo Fisher Scientific). Each sample was analyzed in technical triplicate, and the absorbance values were normalized to sample blanks in which DPP--IV was replaced with Tris-HCl buffer (0.1 M, pH=8.0; Solarbio, Beijing, PR China). The negative control (no DPP-IV activity) and positive control (DPP-IV activity with no inhibitor) were prepared by using Tris-HCl buffer (100 mM, pH=8.0; Solarbio) instead of the sample or instead of the DPP-IV solution and the sample, respectively. Diprotin A (Sigma-Aldrich) was used as a standard inhibitor. The DPP-IV inhibition rate was calculated using Eq. 3.